Human papillomavirus (HPV) has a circular double-stranded genome that is about kbp in length. The genome of all HPV types contains open reading frames (ORFs), which are DNA regions coding for proteins having similar properties, and is divided into two major regions: early (E) and late (L) regions. The early region of about 4.5 kbp codes for genes which are associated with functions including viral DNA replication (E1), induction or suppression of the action of DNA encoding a protein inducing malignant transformation of host cells (E2), the synthesis of proteins responsible for the growth of host cells and viruses (E4), stimulation of the activity of epidermal growth factor (EGF) and colony stimulator factor (CSF) receptors (E5), and malignant transformation through the permanent survival of cells, activation of oncogenes, and inactivation of tumor suppressor genes (E7). In particular, the oncogenic E6 and E7 proteins, which are expressed after HPV infects the epithelial cells of a host, bind to the tumor suppressor proteins of host cells, p53 and pRB, respectively, and thus inhibit the function of the tumor suppressor proteins, resulting in the neoplastic transformation of infected cells.
The late region of 2.5 kbp comprises genes coding for viral major (L1) and minor (L2) capsid proteins and a non-coding region 1 kbp long, which is called the long control region (LCR), and regulates the transcription and translation of the two late genes.
With recent rapid advances in molecular biological techniques, the genetic structure of HPV has been identified, and thus, the genomic sequences of many HPV genotypes were revealed. HPV is classified according to the difference in DNA sequences of E6, E7 and L1 ORFs. When the nucleotide sequences of the ORFs differ by more than 10%, an HPV is assigned a new genotype. HPV subtypes differ by 2% to 10%, and HPV variants differ by less than 2%. To date, over 120 types of HPV have been identified according to the classification.
HPV has been associated with anogenital cancer, laryngeal cancer and tongue cancer. HPV has also been considered a necessary factor for the development and persistence of cervical cancer. Cervical cancer, a malignant tumor that occurs in tissues of the cervix, accounts for more than 95% of all uterine cancers. Worldwide, cervical cancer is the second most common cancer in women after breast cancer, and about 44,000 new cases are reported each year.
Certain types of HPV are classified as “high-risk” because they have high potential for progression to cancer, including cervical cancer. High-risk HPV types include 16, 18, 26, 30, 31, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 61, 66, 67, 68, 69, 70, and 73. Other types, such as HPV 2, 3, 6, 7, 10, 13, 32, 40, 42, 43, 44, 55, 54 and 57, are categorized as “low-risk” because they have lower potential for malignancy.
Due to the close relationship between HPV infection and cervical cancer development and the high death rates of cervical cancer, various strategies have been designed to develop effective vaccines against HPV for the prevention and treatment of cervical cancer.
Prophylactic vaccines, which are given before exposure to HPV, induce the generation of virus neutralizing antibodies, and thus prevent mucosal HPV infection. Therapeutic vaccines induce a cell-mediated immune response targeted against epithelial cells from persons infected with HPV. Therapeutic vaccines eliminate cells expressing the late genes when administered upon viral replication, and, when administered upon the integration of viral DNA into a host cell genome, target E6 and E7 oncoproteins and thus control or suppress the growth of existing HPV-associated tumors. Thus, prophylactic vaccines should be administered prior to HPV infection, while therapeutic vaccines should be administered when lesions are generated by infection or HPV. With respect to action targets according to the molecular biological mechanism of HPV infection, prophylactic vaccines target the capsid L1 or L2 proteins and induce neutralizing antibodies thereto, while most therapeutic vaccines are intended to stimulate the immune system against E6 or E7 early antigens.
Prophylactic vaccines are disadvantageous in that the use of prophylactic vaccines alone cannot treat HPV that has infected cells in the basal layer and has already transformed cells. Prophylactic vaccines induce antigen-antibody responses, but established infections cannot be treated via humoral immunity, but can be treated only via cellular immunity. The therapeutic limitation of prophylacticvaccines has driven the development of therapeutic vaccines. Most therapeutic vaccines target the viral E6 and E7 proteins, which are consistently retained and expressed in cells of the basal layer and cells that have already transformed.
Since premalignant lesions usually contain fewer tumor cells than invasive malignancy, immune responses induced in early lesions may eradicate tumor cells more effectively. In addition, after the early tumorigenesis' stage, MHC class I and II are expressed at lower levels, which could hamper the presentation of tumor antigens, leading to decreased immune responses. Some studies have reported that the lymphocyte proliferation responses to HPV-16 E5 are inversely proportional to the severity of the squamous intraepithelial neoplasia lesions (SILs). Hence, in E5-expressed precancerous lesions, such as SILs and condyloma, using E5 as a vaccine target to induce cytotoxic T cell (CTL) activity specific to E5, which is expressed in earlier stages of HPV infection, may be a good strategy to prevent premalignant lesions from progressing into invasive cervical cancers.
In addition, the L1 major capsid protein, which is produced during the late stage of HPV infection and assembles the replicated HPV genomic DNA into infectious virions in terminally differentiated epithelial cells, has been repotted to induce effectively humoral and cellular immune responses. Hence, the use of the E5 gene along with the L1 gene offers effective treatment effects by inducing L1-specific cellular immunity during all stages of viral infection.
In this regard, the present inventors intended to develop a therapeutic HPV vaccine that targets E5 and effectively eradicates tumors in earlier stages. Also, the present inventors intended to develop a combination DNA vaccine having prophylactic effects as well as therapeutic effects during all stages of viral infection by adding, to the above vaccine, a gene encoding an L1 and/or L2 capsid protein of HPV. As a result, a DNA vaccine comprising both an HPV E5 gene and an HPV L1 and/or an L2 gene was found to induce effectively humoral and cellular immune responses. In addition, when the vaccine was injected intradermally using a tattoo device, it showed remarkably increased immune responses even with very low amounts of antigens and in a short period of time, thereby leading to the present invention.